Project Summary The objective of this proposal is to develop next-generation isobaric labeling reagents that will greatly expand the utility of multiplexed quantitative proteomics and its applications in clinical and pre-clinical biomedical research. The isobaric labeling strategy involves a set of chemical reagents for tagging peptides and other biomolecules with a stable isotope-encoded barcode. The ability to mix and analyze multiple barcoded samples simultaneously without increasing LC-MS complexity. This preserves analytical depth, reduces experimental variability, and increases throughput. This technology is extending the boundaries of proteomics into more profound areas of primary and clinical research, with incredibly powerful and far-ranging applications reported in areas such as drug target identification, biomarker discovery, and temporal regulation of proteome dynamics. While isobaric labeling has been established as an accurate, reliable, and sensitive quantitative technique, there is a definitive need for improvement in isobaric multiplexing capacity. The 11-fold multiplexing of current isobaric labeling reagents limits analysis to relatively simple experimental designs, and is insufficient for the large-cohort analyses required to achieve biological insight in the context diversity among the human population. The reagents proposed herein employ a novel mechanism that involves spontaneous formation of multiple barcodes per reagent, providing the ability to encode additional information, via heavy isotopes, which ultimately expands the multiplexing capacity these reagents for quantitative proteomics. This proposal will address key remaining hurdles to commercialization of these reagents by 1) increasing reagent performance through the development of optimized LC-MS methods, 2) refining data analysis methods to improve measurement precision, 3) developing turnkey software for seamless integration of the approach into current proteomics workflows, 4) using computer modeling to establish an optimal prototype 20-Plex reagent set, 5) synthesis and validation of this prototype reagent set across a variety of applications, and 6) establish a synthesis strategy that is scalable to commercial demand.